Association between colonization of the respiratory tract with Ureaplasma species and bronchopulmonary dysplasia in newborns with extremely low gestational age: a retrospective study

Aim To ascertain the incidence of respiratory tract colonization in extremely low gestational age newborns (ELGANs) with Ureaplasma parvum and Ureaplasma urealyticum and determine if there is a difference in the severity of bronchopulmonary dysplasia (BPD) between ELGANs with and without Ureaplasma species (spp) colonization. Methods We reviewed the medical records of ELGANs 23 0/7–27 6/7 weeks of gestation, tested for U. parvum and U. urealyticum in our Center from January 1, 2009 to December 31, 2019. Ureaplasma spp were identified with the Mycofast Screening Revolution assay based on liquid broth cultures or with polymerase chain reaction. Results This study enrolled 196 preterm newborns. Fifty (25.5%) newborns had Ureaplasma spp respiratory tract colonization, with U. parvum being the predominant species. The incidence rate of respiratory tract colonization with Ureaplasma spp slightly increased in the studied period. The incidence rate for 2019 was 16.2 per 100 infants. BPD severity significantly correlated with Ureaplasma spp colonization (P = 0.041). After controlling for other risk factors for BPD in a regression model, preterm infants colonized with Ureaplasma spp had 4.32 times (95% confidence interval, CI 1.20-15.49) higher odds for developing moderate-to-severe BPD. Conclusions U. parvum and U. urealyticum could be associated with the development of BPD in ELGANs.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease affecting premature neonates and a major cause of morbidity in extremely low gestational age newborns (EL-GANs; gestational age <28 weeks). The pathogenesis of BPD is complex and influenced by both prenatal and postnatal factors (1)(2)(3). Low gestational age (GA) and low birth weight, both of which reflect severe lung immaturity, are inversely correlated with the risk of developing BPD (1,2).
Ureaplasma species (spp), consisting of Ureaplasma parvum (serovars 1, 3,6,14) and Ureaplasma urealyticum (serovars 2, 4, 5, 7-13), has frequently been isolated in amniotic fluid, samples from cord blood, and respiratory tract samples from preterm infants who later developed BPD (3). Ureaplasma spp is a part of normal vaginal flora in 40%-80% of healthy, asymptomatic women, but its presence in the reproductive tract has been causally linked to chorioamnionitis, preterm delivery, miscarriage, and neonatal morbidity (4)(5)(6)(7)(8)(9). The most common route of newborn infection is thus during passage through the colonized vaginal canal. The pathogenetic role of Ureaplasma spp in the development of BPD is controversial, with approaches to its detection and treatment differing greatly among European neonatal intensive care units (10). The existing studies on this issue have been difficult to interpret due to different sample sizes, inconclusive results, and differences in inclusion and diagnostic criteria for BPD (3,11).
Meta-analyses (12,13) reported higher odds for BPD development in infants colonized with Ureaplasma spp. Studies included in these meta-analyses used the original definition of BPD as oxygen dependence at 28 days of life (12,13). This definition has later been updated to reflect the increased survival of extremely premature infants that often need supplemental oxygen or respiratory support in the first weeks simply due to lung immaturity (14).
A growing body of evidence from both experimental models and immunological studies, due to a better understanding of virulence factors and host-pathogen interactions, supports the causal role of Ureaplasma spp colonization in BPD development (15)(16)(17). Respiratory tract colonization with Ureaplasma spp has proinflammatory and profibrogenic effects and thus contributes to the development of BPD either alone or in combination with inflammatory factors such as hyperoxia or mechanical ventilation (16). However, the impact of Ureaplasma-driven inflammation on neonatal morbidity has been controversial, and the clinical relevance of detecting Ureaplasma spp in preterm neonates remains a subject of discussion (18).
The present study was conducted in a tertiary perinatal center where the incidence of BPD has been slightly increasing in recent years despite the fact that we followed recommendations for BPD prevention, including the use of non-invasive and protective ventilation. The aim of this study was to ascertain the incidence of respiratory tract colonization in ELGANs with U. parvum and U. urealyticum and to determine whether early-life colonization with Ureaplasma spp is associated with the development and severity of BPD in our group of ELGANs.

Patients
This retrospective study enrolled preterm infants with extremely low GA from 23 0/7 to 27 6/7 weeks hospitalized at the Neonatal Intensive Care and Therapy Unit (EINT), Neonatology Section, Department of Perinatology, Division of Gynecology, University Medical Centre Ljubljana in the period from January 1, 2009 to December 31, 2019, who were tested for U. parvum and U. urealyticum. We excluded all infants who died within 24 hours after birth. We collected pregnancy and perinatal data, and the data on treatment course and morbidities.
Maternal and newborn characteristics were chosen based on previous studies (19). The following prenatal maternal data were collected: age, parity, type of labor, administration of prenatal steroids and prenatal antibiotics, diagnosis of diabetes with insulin treatment, primary hypertension, chorioamnionitis and/or preeclampsia, or eclampsia.
The newborns' data included the number of days spent in EINT, inborn or outborn delivery, death, GA, birth weight, small for GA, type of resuscitation in the delivery suite, timing of surfactant application, fraction of inspired oxygen (FiO 2 ) when surfactant was administered, method of surfactant delivery (less invasive surfactant administration; minimally invasive surfactant therapy; intubation-surfactantextubation; invasive application with longer intubation), the number of surfactant applications, the use of postnatal steroids, duration of noninvasive ventilation (continuous positive airway pressure; nasal intermittent positive pressure ventilation; high-flow nasal cannula), duration of invasive mechanical ventilation or high-frequency oscillation (HFO) ventilation, duration of iNO respiratory support, the number of days with FiO 2 >0.21, and the number of days when there was a need for respiratory support (immediately after birth or after 2-3 weeks) and vasopressor use.
Additional information on the incidence of BPD was obtained from the Vermont Oxford Network.
Newborn-associated morbidities included stages of BPD, pneumothorax, necrotizing enterocolitis, early or late sepsis, systemic inflammatory response syndrome, or any other infections (cytomegalovirus infection; pneumonia or both), U. parvum or U. urealyticum infection, and the use of azithromycin.
To calculate the incidence of Ureaplasma spp colonization of ELGANs in EINT we also collected the number of all EL-GANs treated in our institution for each year. The research was approved by the National Medical Ethics Committee of Slovenia.

Classification of BPD
We used the BPD definition proposed by the National Heart, Lung, and Blood Institute (NHLBI) Workshop criteria (20). Three levels of disease severity are used for preterm infants with GA of less than 32 weeks: 1) mild BPD, defined as a requirement for at least 28 days of supplemental oxygen therapy and discharge or termination of supplemental oxygen therapy by 36 weeks postmenstruation age; 2) moderate BPD, defined as a requirement for at least 28 days of supplemental oxygen therapy with FiO 2 less than 0.3 at 36 weeks postmenstruation age; and 3) severe BPD, defined as a requirement for at least 28 days of supplemental oxygen therapy with FiO 2 0.3 or greater at 36 weeks postmenstruation age (20). Patients were additionally divided into two groups: 1) those without BPD or with mild BPD and 2) those with moderate or severe BPD. Because the used criteria do not define the severity of BPD for newborns who die before 28 days of life, we subsequently excluded additional 5 newborns. For three newborns that died between 28 days and 36 weeks postmenstrual age we determined BPD severity based on the need for supplemental oxygen in the last days before death. The incidence of respiratory tract colonization of ELGans with Ureaplasma spp The incidence rate of respiratory tract colonization with Ureaplasma spp slightly increased in the study period (Figure 1). Interestingly, BPD incidence also increased in the study period, from 17% in 2009 to 30% in 2019 ( Figure 2). The average incidence rate of respiratory tract colonization with Ureaplasma spp was 8.8 per 100 newborns per year.
The average day of testing for Ureaplasma spp colonization was the 23th day of life.

Prenatal maternal characteristics and BPD
The univariate logistic regression analysis showed no significant association between prenatal maternal characteristics and the severity of BPD (Table 1).

newborn characteristics and BPD
The likelihood ratio test showed a significant association between BPD severity and death of newborns (P = 0.013) and between BPD severity and colonization with Ureaplasma spp (P = 0.041, Table 2). After controlling for other previously known risk factors for moderate-to-severe BPD, multiple logistic regression analysis showed that preterm infants colonized with Ureaplasma spp had 4.32 times (95% confidence interval, CI 1.20-15.49) higher odds for moderate-tosevere BPD (Table 3).

DisCUssion
In this study, the development of moderate-to-severe BPD in ELGANs was significantly associated with Ureaplasma spp colonization. Unlike similar studies that mostly screened infants for Ureaplasma spp colonization soon after birth, this study enrolled infants that had their microbiological samples taken later during treatment due to a clinically suspected infection or developing BPD. Thus, 95% of the infants included in this study developed BPD according to the NHLBI diagnostic criteria. There was an important clinical and prognostic distinction between the group without or with mild BPD and the group with moderate or severe BPD, with the group without or with mild BPD having better lung function and better spirometry results (21).
Our findings agree with the findings of a meta-analysis by Schelonka et al (22), who reported a significant association between Ureaplasma spp colonization and BPD36 development (22). BPD36 corresponded to moderate and severe BPD according to the NHLBI criteria. However, an-other meta-analysis, including studies that only looked at colonization with U. urealyticum, did not find an association with BPD development (23). Our study did not find a difference in BPD development between U. parvum-and U. urealyticum-colonized infants; however, U. parvum colonization was three times as frequent (36 vs 12 infants). In the study by Glaser (24). Similarly, the risk for BPD increased in Ureaplasma-colonized infants that were mechanically ventilated for five days or more, but the colonization itself was not associated with a higher risk for BPD (18).
This study found an increasing incidence of U. parvum and U. urealyticum colonization among ELGANs in Slovenia's largest tertiary perinatal center in the period 2009-2019, and a lower average incidence compared with other studies. Interestingly, we also found an increase in the incidence of BPD in ELGANs in the study period, which correlates with the increased incidence of Ureaplasma coloniza- tion. In a study that used PCR as the method of detection, respiratory tract colonization with Ureaplasma spp in infants with very low birth weight (<1500 g) was 25%-48% (2). In another study, the rate in newborns younger than 26 weeks GA was 65% (25). In a more recent trial, the rate in infants younger than 29 weeks GA was 36% at one or more time points (16). In our study, the average incidence of respiratory tract colonization with Ureaplasma spp among ELGANs was only 8.8%.
A possible explanation for the differences between ours and other studies is a slightly different population of tested infants and a later sample collection. The mentioned studies collected microbiological samples aged <72 hours from all infants with GA younger than 26 or 29 weeks. In our study, the microbiological samples were collected later during treatment and only from infants that were clinically suspicious for developing BPD or signs of infection. The average time of testing for Ureaplasma spp in our institution was the 23th day after birth. In a study by Payne et al (26), the highest detection rate of U. parvum in aspirates from ELGANs was found 3-5 days after birth. It is worth noting, however, that three patterns of U. urealyticum colonization have been reported in preterm infants: persistent, early transient, and late transient. Only the persistently positive colonization pattern was associated with a significantly increased risk of developing BPD (27). It is thus possible that screening all ELGANs soon after birth would have led to a higher incidence of colonization in our institution as well. Finally, the incidence of Ureaplasma spp colonization among ELGANs and/or women of childbearing age could be lower in our geographical area, but we could not find any studies investigating this issue.  be the introduction of PCR in the third year of this 11-year study, which is more sensitive than culture as a method of detection (28).
A strength of this study is the data collection from a relatively large cohort of infants over a long, 11-year period. We included infants treated in Slovenia's largest tertiary perinatal center, where 80% or more of all ELGANs receive intensive care. Our medical team used the same criteria and standards throughout the study period, which allowed us to reliably compare the year-by-year results.
This study has several limitations. First, the retrospective design prevented us from controlling the exact indications for or the timing of Ureaplasma spp testing and other variables. The decision to collect microbiological samples was made by the treating physician. Another limitation were the NHLBI diagnostic criteria themselves, as we could not include infants that died before the 28th day of age. Additionally, we could not use the recently proposed BPD severity diagnostic criteria that include x-ray imaging, as it was not routinely performed in our institution (29). Finally, one possible confounding factor could be the replacement of culture as the method of microbiological analysis with PCR, which is a more sensitive method that could have detected the pathogens that culture missed.
In conclusion, this study found an increasing incidence rate of Ureaplasma spp airway colonization in ELGANs treated in our institution in the period 2009-2019. This finding could in part be attributed to the greater proportion of tested infants and the greater sensitivity of microbiological methods. Also, BPD severity was significantly associated with the colonization with Ureaplasma spp. After we controlled for other risk factors for BPD in the regression model, preterm infants colonized with Ureaplasma spp at the average age of 23 days had 4.32-times higher odds for developing moderate or severe BPD. Screening all ELGANs soon after birth and early treatment of Ureaplasma spp colonization could reduce BPD incidence.
Funding None.
Declaration of authorship ŠG and TP conceived and designed the study; KG and RM acquired the data; DK, VE, ŠG, and TP analyzed and interpreted the data; KG and RM drafted the manuscript; DK, VE, ŠG, and TP critically revised the manuscript for important intellectual content; all authors gave approval of the version to be submitted; all authors agree to be accountable for all aspects of the work.
Competing interests All authors have completed the Unified Competing Interest form at www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare: no support from any organization for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years; no other relationships or activities that could appear to have influenced the submitted work.